(Adapted from the Applicant's Abstract) The long-range goal of this project is to further the understanding of the genetic regulation of cardiac development by elucidating the function of the Cardiac-specific homeobox Csx/Nkappax-2.5 protein. In the mouse this homeobox protein is expressed in the heart throughout the heart's transition from pluripotent pre-cardiac mesoderm cells to structural maturity. Mice homozygous for a mutation for a mutation for a mutation for these gene are embryonic. Mice homozygous for a mutation for this gene are embryonic lethal due to an arrest in cardiac development at the looping stage prior to septation, Tanaka and Izumor, unpublished). Previous studies have demonstrated that, like other homeobox proteins, Csx/Nkappax2.5 is a DNA-binding protein which can transactivate expression from specific promoter sequences and associates with other transcription in vitro. Due to the severe phenotype of mice lacking the Csx/Nkappax2.5 play a critical role in directing normal myocardial growth and development. The continued expression of this gene throughout the heart's structural development and in the adult heart suggests that the target genes of the Csx protein may also be fundamental to the maintenance of the cardiac muscle phenotype post-natally. The investigation of the transcription factor cascade that controls cardiac structural growth and development in the mouse may yield important insights into the genesis of congenital heart diseases. The key elements of this cascade are conserved from invertebrates, such as Drosophila, to vertebrates, such as chicken and mice. In prior studies in the laboratory, both the mouse Csx/Nkappax2.5 gene and its human homologue were identified and determined to be highly homologous (100 percent amino acid identify within the homeodomain, TN domain and Nk2 domain). This, together with the high degree of conservation of the genetic cascade directing cardiac organogenesis, suggests that the studies Csx/Nkappax2.5 protein function and its downstream targets are likely to yield important insights into the molecular basis of congenital heart disease in humans. Accordingly, the investigators will address the following specific aims in this proposal. Specific Aim 1: To fully characterize the phenotype of homozygous and heterozygous Csx/Nkappax2.5 null mice by molecular biological, physiological and morphological analyses. Specific Aim 2: To determine structure-function relationships of Csx/Nkappax2.5 in vitro by DNA binding, and transcriptional activation assays by mutating the highly conserved TN domain, the NK2-specific domain, and CD-II phosphorylation sites. Specific Aim 3: To determine the biological function of the TN- domain, the NK-2 specific domain and CKII phosphorylation site of Csx- Nkappax2.5 using gene targeting with Cre-loxP mediated excision. Specific Aim 4: To create mutations in murine Csx/Nkappax2.5 in the analogous positions found in patients with ASD associated with AV conduction block and (a) to assay their DNA binding, transactivation and protein-protein interaction function in vitro, and (b) to create mouse models of congenital ASD associated with conduction block by gene-targeting technique of congenital ASD associated with conduction block by gene targeting technique and study their morphological, functional and electrophysiological abnormalities. Specific Aim 5: To identify transcriptional targets of Csx/Nkappax2.5 by combing the methods of the yeast one-hybrid screening of a Csx-target enriched genomic DNA library and differential display of double homozygous Csx/N kappax2.5 null ES cells that harbor a tetracycline responsive Csx/Nkappax2.5 transgene.